Tumor necrosis factor alpha gene variants do not display allelic imbalance in circulating myeloid cells

Cellular Immunology 262 (2010) 127–133

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Tumor necrosis factor alpha gene variants do not display allelic imbalance in circulating myeloid cells Sandra Wienzek *,1, Karin Kissel 1, Kirstin Breithaupt, Christina Lang, Angelika Nockher, Holger Hackstein, Gregor Bein Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University Giessen, Giessen, Germany

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Article history: Received 30 October 2009 Accepted 4 February 2010 Available online 10 February 2010 Keywords: Tumor necrosis factor a Genetic polymorphism Risk allele TNF 308 A/G Allelic imbalance

a b s t r a c t Carriage of the TNF 308 A allele (rs1800629 A) has been associated with increased serum TNF-a levels, the development of sepsis syndrome, and fatal outcome, in severely traumatized patients (Menges et al., 2008 [1]). Herein, we analysed the putative allelic imbalance of TNF-a release from myeloid cells. Circulating peripheral blood cells from healthy human blood donors (n = 104) and monocyte-derived macrophages (n = 158) were analysed for their ex vivo capacity of TNF-a expression. Our findings indicate that carriage of the TNF 308 A allele is not associated with high TNF-a expression in circulating human leucocytes and monocyte-derived macrophages. Other cellular sources, e.g. tissue-resident cells like mast cells and/or tissue specific macrophages might be the cellular source of high TNF-a serum levels shortly after trauma. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Tumor necrosis factor alpha (TNF-a) [2] is an inflammatory cytokine expressed in a variety of cell types like macrophages, monocytes and neutrophils [3–8]. Analysis of TNF-a levels in the blood of healthy individuals shows broad but reproducible interindividual variation [9,10], indicating that there is a genetic basis determining its expression. Within the TNF promoter region, numerous single nucleotide polymorphisms (SNPs) are located [11–17]. TNF promoter variant 308 G>A has been associated with several sepsis related phenotypes [1,18,19]. The association of the TNF 308 G>A polymorphism with systemic endotoxin-triggered inflammation has been studied with controversial findings [20–27]. Recently, Menges and colleagues [1] reported replicated association between TNF haplotypes (haplotype SNPs, TNF 308 A and LTA +252 G) and high TNF- a serum levels, sepsis syndrome and death in trauma patients. This and other studies [18,19] suggest that TNF haplotypes display allelic imbalance of TNF-a release upon stimulation and that the TNF 308 A allele might represent a high responder allele leading to hyperinflammation and susceptibility to sepsis in carriers of one or two copies of this allele. Due to the fact that only rare data are available from studies examining

* Corresponding author. Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University Giessen, Langhansstrasse 7, D 35392 Giessen, Germany. Fax: +49 641 41509. E-mail addresses: [email protected] (S. Wienzek), [email protected] (K. Kissel). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.02.004

TNF-a cytokine release from monocyte-derived macrophages and peripheral circulating blood cells, our study addressed this question using large cohorts of healthy individuals. We utilized positive and negative cell sorting to examine TNF-a releasing capacity upon LPS and IFN-c challenge from different peripheral blood cells (natural killer cells, neutrophils, monocytes), and macrophages derived from CD14+ monocytes. Genotyping was performed by TaqMan allelic discrimination. In statistical analysis, an association between TNF-a release and the TNF promoter polymorphism 308 G>A was explored. 2. Materials and methods 2.1. Characteristics of the individuals In the current prospective study, blood samples of healthy male and female donors between 18 and 45 years were randomly selected. Donor’s consent was supplied by each individual blood donation. The study was approved by the Ethic Study Board of the University Hospital of Giessen (file number: 05/00). 2.2. Isolation and stimulation of circulating blood mononuclear and polynuclear cells Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by Ficoll–Hypaque (d: 1.077 g/cm3 Pharmacia, Freiburg, Germany) density centrifugation according to manufacturer’s instructions.

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Human neutrophils were isolated from EDTA-anti-coagulated blood of healthy donors by dextran sedimentation and gradient centrifugation (Ficoll–Hypaque) for 20 min at 500g. Contaminating red cells were lysed by adding PuregeneTM-RBC-Lysis Solution (Gentra Systems, Minneapolis, MN, USA). Experiments to remove CD16+ and CD56+ cells were performed using magnetic beads selection (Miltenyi Biotec, Bergisch Gladbach, Germany) on AutoMACS (Miltenyi Biotec) according to manufacturer’s instructions. CD14+ monocytes were positively selected by the use of CD14+ coupled immuno magnetic particles (Miltenyi Biotec) on AutoMACS (Miltenyi Biotec) as mentioned by the company. Purity of monocyte preparations were proved by cell counting (Sysmex-F 820, Norderstedt, Germany; level of contaminating platelets below the detection limit (<1  103/ll) and on the basis of cell morphology. For generation of monocyte-derived macrophages, 1  105 CD14+ cells were expanded in 96-well flat bottom plates containing 0.2 ml medium consisting of RPMI 1640 with L-glutamine (PAA Laboratories, Pasching, Austria), 0.5% penicillin/streptomycin (10000 U/ml, Bayer, Leverkusen, Germany), and 10% FCS (PAA Laboratories, Linz, Austria). To induce differentiation into macrophages, 50 ng/ml macrophage-colony stimulating factor (M-CSF, Promokine, Heidelberg, Germany) was supplemented and cells were cultered for 4 days at 37 °C, 5% CO2 atmosphere [28]. Control of macrophage morphology was performed using 1  105 cells cultured for 4 days in M-CSF on the basis of spindle-like morphology and cell adherence by light microscopy (Leica DM IRB, Wetzlar, Germany). 2.3. Staining and flow cytometry Macrophages phenotypical analysis was performed on day 4. Cells (3  106) were washed with 3 ml PBS twice each and removed from plastic plates by treating with Trypsin–EDTA (0.5% Trypsin, 0.2% EDTA, GIBCO BRL, Eggenstein, Germany) for 20 min. Trypsin– EDTA was removed by adding 12 ml medium. Subsequently, cells were pelleted (250 g, 5 min), resuspended in staining buffer (SB; BD Cytofix/Cytoperm TM Plus Fixation/Permeabilization Kit, BD Biosciences) and counted. Macrophages were labelled for CD68, CD1a and CD14 expression using BD Cytofix/Cytoperm TM Plus Fixation/ Permeabilization Kit (BD Biosciences, San Jose, CA, USA) according to company’s instructions. Neutrophils were phenotyped in by the use of FITC-labelled CD16 (clone 3G8). Detection of intracellular TNF-a was performed on BD Cytofix/Cytoperm TM Plus Fixation/ Permeabilization Kit (BD Biosciences) either in the presence of Brefeldin A (1 lg/ml, GolgiPlug) or Monensin (6 lg/ml, GolgiStop) using TNF-a clone MAb 11. All mabs, including isotype controls, were purchased from BD Biosciences. Phenotypic analysis of cells was performed on FACSCalibur (BD Biosiences). 2.4. Measurement of cytokine production To perform TLR stimulation experiments, freshly isolated PBMCs (5  105) from the blood of healthy donors were incubated with TLR ligands HKLM, Poly I:C, LPS, Pam3CSK4, Flagellin 1, FSL1, Imiquimod, ssRNA and ODN (Human TLR 1-9 Agonist Kit, Invivogen, San Diego, USA) in concentrations according to manufacturers instructions. For the large cohort study, 1  105 cells were stimulated on day 4 (macrophages) or day 0 (PBMCs) by adding a cocktail containing 10 ng/ml IFN-c and 1 lg/ml LPS strain 02:B6 (Sigma–Aldrich, Munich, Germany), or LPS (1 lg/ml) alone. Optimal concentrations of LPS and IFN-c were determined by titration experiments. To achieve control experiments, cells were incubated without stimuli. Supernatants were harvested at distinct time points depending on experiment (PBMC: 8 and 18 h; macrophages: 1 h; time course experiments: 2, 4, 8, 18, 24 h) and cytokine production was mea-

sured using ELISA technique (BD OptEIA ELISA sets, BD Biosciences) according to manufacturer’s instructions. All stimulation experiments were run in duplicates, and the mean was used as outcome value. For measurement of cytokine production upon crosslinking of CD32a, 100 ll of IgG solution (1 mg/ml) (Gamunex Bayer Vital, Germany) was coated onto microtiterplates (18–20 h; 4 °C). After four wash steps using 200 ll PBS each, cells were added and treated as described above. Detection of cytokine production on single cell basis was performed using ELISPOT analysis according to manufacturer’s instructions. Spots were analysed on a Bioreader-400 (BioSys GmbH, Karben, Germany). 2.5. Genotype analysis Genotyping was performed using TaqMan allelic discrimination using the Assay-by-SNP genotyping assays (Applied Biosystems, Foster City, CA, USA). PCR was carried out using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) according to company’s protocol and using sequence-specific primers for SNP rs 1800629 (1). Accuracy of genotyping was confirmed by sequencing of reference DNA samples. Negative controls for each run were performed using water samples. 2.6. Statistical analysis Association of TNF promoter polymorphism 308 G>A with TNF-

a release was calculated using two-tailed Mann–Whitney-U-test (SPSS software version 12.0.1, SPSS Inc. Chicago, Il). Wilcoxon-test was used in analysis of TRL-ligands. Rare genotype variants TNF 308 GA and TNF 308 AA were combined in one group. 3. Results 3.1. Among PBMCs, CD14+ CD16+ monocytes are the main source of TNF-a To get more insight into the capacity of different subsets of peripheral blood mononuclear cells (PBMCs) to release TNF-a, we treated freshly isolated PBMCs of male blood donors with different Toll like receptor (TLR) ligands. Fig. 1 demonstrates that LPS, HKLM, and Poly I: C are capable to induce high amounts of TNFa. Using Pam3CSK4, Flagellin 1, FSL1, Imiquimod, ssRNA and ODN, no significant TNF-a release was found. It was also noted, that synergistic effects of IFN-c and LPS became obvious in our kinetic study (Fig. 2). A maximal concentration of TNF-a upon stimulation with LPS was observed after 8 h, whereas the maximal concentration of TNF-a upon simultaneous stimulation with LPS and IFN-c was observed after 18 h. After this time period, TNF-a level decreased. The most remarkable finding was that removal of CD16+ cells resulted in significantly decreased levels of TNF-a, as demonstrated in Fig. 1. The same TNF-a levels were detected before and after depletion of CD56+ cells (data not shown). Thus, CD14+CD16+ monocytes, but not CD16+CD56+ natural killer cells are the main source of TNF-a in PBMCs. 3.2. TNF-a release from human monocytes is not affected by carrying TNF 308 A To answer the question whether TNF-a levels derived from CD14+CD16+ monocytes might be influenced by the presence of the rare TNF 308 A allele, PBMCs from a large cohort of 104 healthy donors were analysed. Although we identified interindividual variation of TNF-a levels, an association between TNF 308 G>A and TNF-a release was not detected (p > 0.05; Mann–

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Fig. 1. TNFa release from CD16-depleted and untouched PBMCs upon stimulation with TLR-ligands. Human PBMCs or CD16+ depleted PBMCs (n = 7) were cultured in the presence or absence of TLR ligands 1–9 for 18 h. Supernatants were examined for TNF-a levels using ELISA. LPS revealed as the most potent inducer of TNF-a release. In the presence of LPS, the production of TNF-a was increased 6.7-fold, HKLM: 6.3-fold, and with Poly I:C 5.5-fold compared to non-stimulated PBMC. Depletion of CD16+ cells resulted in reduced TNF-a levels. Mean values and standard errors were shown above.

unstimulated LPS 1 µg/ml

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rophages from 158 healthy individuals were investigated. We found that the TNF-a release from macrophages upon stimulation was not significantly elevated in carriers of the TNF 308 A allele (p > 0.05; Mann–Whitney-U-test), as demonstrated in Fig. 4.

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3.4. Human circulating neutrophils produce only rare amounts of TNF-a

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Fig. 2. Kinetics of TNF-a release from PBMCs upon stimulation with LPS and IFN-c. Human PBMCs (5  105) were stimulated in time course experiments for 2, 4, 8, 18, 24 or 48 h with or without LPS 1 lg/ml, IFN-c 200 U/ml and a cocktail containing LPS and IFN-c. Supernatants were examined for TNF-a levels using ELISA. Highest amounts of TNF-a could be detected within 8 and 18 h (LPS and LPS plus IFN-c, respectively). LPS and IFN-c showed additive (synergistic) effects.

Whitney-U-test), as demonstrated in Fig. 3a. Cross-linking of Fcc receptor IIa (CD32a) by complexed IgG resulted in increased TNFa levels, but activation of CD32a did not reveal an association between high cytokine levels and carriage of the TNF allele 308 A (p > 0.05, Mann–Whitney-U-test; Fig. 3b). 3.3. The TNF 308 A allele is not associated with higher TNF-a release from monocyte-derived macrophages Next, we hypothesised that tissue-resident cells like macrophages are putative candidates for the manifestation of allelic imbalance. To test this assumption, CD14+ cells were separated from peripheral blood and treated with M-CSF. Cultured cells underwent change characteristics of monocyte differentiation into macrophages such as spindle-like morphology and increased adherence (data not shown), as has been described for in vitro generated macrophages [29,30]. Monocyte differentiation was also mirrored by changes in cellular marker proteins CD14 and CD68, revealing macrophage-like phenotype in FACS analysis (data not shown). To test the association of TNF 308 A allele with the quantitative phenotype TNF-a release in vitro, monocyte-derived mac-

To answer the question whether circulating neutrophils might be a source of significant amounts of TNF-a, we stimulated them either with LPS, LPS plus IFN-c or left them unstimulated. TNF-a was not detectable neither intracellular nor on the cell surface of CD16+ neutrophils using FACS analysis (data not shown). The amount of secreted TNF-a upon stimulation was low compared to PBMCs and late (24 h) (ELISA, data not shown). To analyze the TNF-a production in neutrophil suspensions on a single cell level we employed an enzyme linked immuno spot assay [31]. As demonstrated in Fig. 5, removal of CD14+ cells from neutrophil suspension using immuno-magnetic beads resulted drastically reduced amounts of TNF-a producing cells, when compared to untouched fraction. These findings provide evidence that TNF-a in neutrophil suspensions was produced by a few contaminating CD14+ monocytes. There was no evidence for TNF-a release from neutrophils upon stimulation with LPS and/or IFN-c. In summary, analysing large cohorts of healthy blood donors, our results indicate that carriage of the TNF 308 A allele is not associated with the phenotype high TNF-a expression in human circulating monocytes, NK cells, neutrophils, and monocyte-derived macrophages. 3.5. Genotyping results TNF promoter polymorphism rs1800629 ( 308 G>A) was determined using TaqMan technique. The genotype frequencies showed compatibility with Hardy–Weinberg equilibrium. Allele frequency agreed with the published frequency in Caucasians (http:// pga.mbt.washington.edu). 4. Discussion The aim of the present study was to identify the main TNF-a producing cellular subset of circulating peripheral blood cells and

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a

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Fig. 3. Association TNF-a release from human CD16+ monocytes and TNF-genotype. (a) PBMCs of 104 healthy donors were isolated and cells were supplemented with the stimuli mentioned above for a time period of 8 h. Following measuring of cytokine release by ELISA and detection of individual’s genotype by TaqManÒ assay. An association between TNF 308 A rs1800629 and TNF-a phenotype was calculated, but could not be verified (p > 0.05) in statistical analysis (Mann–Whitney-U-test). (b) Donor cells used above were analysed in parallel by the additional application of immobilized IgG. Cells were adjusted to cross-linking of CD32a by complexed IgG. An association between TNF genotype 308 A/G, estimated by TaqManÒ analysis, and TNF-a phenotype was calculated using Mann–Whitney-U-Test. Activation of CD32a did not discover an association between high cytokine levels and existence of the TNF rs1800629 allele A (p > 0.05).

p>0,05 1400

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Fig. 4. Association TNF-a release from macrophages and TNF-a genotype. About 1  105 CD14+ selected monocytes from healthy blood donors (n = 158) were expanded 4 days long in the presence of M-CSF (50 ng/ml). Differentiated macrophages were stimulated for 1 h using LPS (1 lg/ml) and LPS plus IFN-c (1 lg/ml and 10 ng/ml, respectively). TNF-a production was measured by ELISA. An association between TNF genotype 308 A and TNF-a phenotype in macrophages was calculated using Mann– Whitney-U-Test. The association was not significant (p > 0.05).

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untouched

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Fig. 5. TNF-a releasing capacity of circulating neutrophils. Magnetic beads sorted CD14+ cells were eliminated from cell suspension and the remaining fraction (‘‘CD14 depleted cells”) subjected to TNF-a specific ELISPOT analysis. The amount of TNF-a producing cells was reduced in CD14 depleted fraction compared to untouched cells (lanes C + D vs. A + B).

to test whether the TNF 308 A allele is associated with elevated TNF-a release upon stimulation of blood cells ex vivo. We identified CD14+CD16+ monocytes as a main source of TNFa in circulating blood, in accordance with results yielded from others [7,8]. Although representing only 5–10% of peripheral blood leucocytes, these cells may be regarded the principal TNF-a producing circulating blood cell [8]. Secondly, reproducing data from literature [32,3], we identified IFN-c and TLR4 ligand LPS as agents with the highest capacity to induce TNF-a release from PBMCs. Usage of immobilized IgG to induce TNF-a production via Fcc receptor signalling pathway revealed increased TNF-a production [33]. However, several experiments including large cohorts of blood donors did not reveal allelic imbalance of TNF-a production. We did not detect an association of TNF-a release from peripheral blood cells ex vivo and TNF 308 G>A genotypes. Based on the results of our depletion experiments, we supposed that CD16+ cells but not T and B lymphocytes are the main source of TNF-a release in PBMCs. Therefore, we asked in a next step whether in vivo tissue-resident macrophages may be the cellular source of TNF-a in trauma patients and whether macrophages may display allelic imbalance of TNF secretion. According to MacKenzie [34], we found a higher amount of TNF-a released from macrophages compared to CD14+ monocytes. Corresponding to their study, we also recognized changes in cellular marker proteins, e.g. CD14 and CD68 during the differentiation process. However, although the expected inter-individual TNF-a cytokine levels [35,9] became obvious, an association between TNF 308 G>A genotypes and TNF-a release from macrophages was not detected (n = 158). In contrast to our findings are results presented by Kroeger and colleagues [36], performing in vitro reporter gene assays. Using TNF 308 G>A transfected cell line U937, they found different promoter activity of TNF 308 G and A transfectants. These findings might depend on the fact that we used primary cells derived from healthy individuals and analysed their amount of TNF-a release instead of transfected cell lines analysed by reporter gene assay. Further, differences in stimulating agents are given and in vitro DNA binding assays may not accurately reflect ex vivo events. However, several aspects have to be kept in mind. Firstly, we analysed CD14+ monocytes-derived macrophages, but the macrophage lineage itself is heterogeneous [37,38]. Monocyte-derived macrophages

may not reflect features of tissue-resident macrophages in vivo. Secondly, our CD14+ monocytes developed into macrophages using M-CSF. There might be functional heterogeneity in ex vivo generated human monocyte-derived macrophages depending on the inducing factor, as M-CSF or G-MCSF [30]. Thirdly, circulating monocytes are also heterogeneous themselves, giving rise to mature macrophages [37]. Stimulus-specific effects may play a role, although we used potent stimulators of TNF-a in macrophages. It is postulated, that IFN-c and LPS modulate the physiologic action of TNF in macrophages through complex mechanisms [32,39] including effects on transcription itself and on its receptors [40] and post-transcriptional modulations [34]. We studied effects of LPS and IFN-c on circulating neutrophils to secrete TNF-a due to their ability to produce and release immune regulatory cytokines early during infection [4,41,42]. Our experimental data confirm findings from Dubravec [4], demonstrating that circulating neutrophils do not store intracellular preformed TNF-a. We saw rare TNF-a producing cells in neutrophil suspensions by the sensitive ELISPOT assay [31] but suggest that TNF-a was rather produced by a few contaminating CD14+ monocytes than produced by terminally differentiated neutrophils. Our ex vivo study was designed to achieve high statistical power. We recruited 104 healthy individuals donating PBMCs, and the size of our monocyte-derived macrophage cohort was n = 158. In all cases, samples were successfully genotyped. Previous ex vivo studies focussing on the association between TNF- a production and SNP genotype observed an association with TNF 308 A allele [9], performing whole blood assays from 57 healthy individuals. Subjects with healthy/disease profile [43] or patients whole blood assays [44] observed an association with TNF 308 A allele. Other authors presented results analysing whole blood LPS stimulation experiments [22], or measuring circulating TNF-a levels in vivo [45], negating the effect of TNF 308 risk allele A on TNF-a secretion. The impact of the TNF 308 G/A polymorphism on systemic endotoxin-triggered inflammation in vivo has been discussed controversial in numerous candidate gene association studies [20–27]. All these findings lead some authors to question the functional relevance of this SNP [25,46,47,27,48,49]. Depending on study design and experimental strategy, distinct results have been reported, with special regard to the finding that TNF-a expression is regulated on all steps in the pathway from

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DNA via RNA to protein [49]. Transcriptional and post-transcriptional events [50–52,34,53], including mRNA regulation through AU-rich elements (ARE) [54,55], activation of the pro-protein through proteases [56] and further adjustment of bioactivity by membrane-shedded TNF receptors [57] regulate protein secretion. It is also thinkable, that numerous other gene products are involved and each of these might control individual levels of TNF-a [32,49]. Resting or activated status of cells might also influence TNF gene expression [52]. Effects of confounding factors may mask true effects of TNF gene variants [49]. TNF-a polymorphism might be only a marker for other genetic factors that are the real cause for the observed differences in TNF-a concentration [58]. Linkage with other genetic variants might be involved in the regulation of TNF-a [46,47,58]. Correlations between TNF-a production and the extended haplotype HLA A1/B8/DR3 [50] and LTA-NcoI polymorphisms [21] have already been described. Possibly, polymorphisms in the promoter region of TNF (TNF 308 A>G) might alter expression of TNF-a and might induce suboptimal activity of TNF-a in people with GG genotype, as discussed by Azim [59]. In this prospective study of patients with esophageal cancer and development of infections, TNF 308 A allele has been shown to be associated with higher circulating levels of TNF-a Recently, strong association of TNF gene variation 308 G>A and the risk of mortality was postulated by Shalhub [60]. However, reports from in vivo studies of TNF-a response to LPS challenge in healthy volunteers negate an association between TNF-a phenotyp and TNF 308 genotype [61,24,25]. These findings are not easily to interpret in context with the results presented by Menges [1]. The effects of the DNA variant might be highly context specific, depending on the specific cell type and specific stimulus. In addition, ex vivo and in vivo studies might not exactly mirror the conditions of individuals in septic situations. Our results do not mean that the TNF 308 G>A gene variants have no biological function, but they point to the fact that the underlying biological cause, if it exists, will not reveal itself easily. To focus on our own results, we excluded subsets of circulating blood cells to be the cellular source of TNF-a in carriers of the TNF risk allele 308 A. Results yielded from De Jong and colleagues [62] confirm our finding. This group examined a cohort of healthy individuals in nearly equal size to ours (n = 129) and stimulated cells with equal concentrations (LPS 1 lg/ml). But our findings take a step forward, presenting results from cultured monocyte-derived macrophages (n = 158) and particular cell fractions from PBMCs vs. whole blood assays performed by De Jong [62]. In addition, we confirmed our finding not only by TLR-dependent stimulation but also in a TLR4 independent way (IgG, IFN-c). There are hints in literature [36] that tissue-resident cells are primarily involved in TNF-a production when carrying the TNF risk allele 308 A. Tissue-resident macrophages are good candidates. Recently, analysing a cohort including 1321 patients developing invasive meningococcal disease, Read [63] reported that TNF polymorphism 308 A is associated with susceptibility to meningococcal sepsis, but not with lethality. In addition, ex vivo generated macrophages from healthy individuals homozygous for the rare allele AA (n = 6) produced higher concentrations of TNF in response to N. meningitidis or to its LPS. Compared to our study, the authors used different experimental settings, including generation of monocyte-derived macrophages (without M-CSF vs. usage of M-CSF), the time point of investigation (12 days vs. 4 days), and the mode of stimulation (usage of N. meningitiditis and its LPS vs. LPS plus IFN-c). In addition to tissue-resident macrophages, mast cells represent a second important tissue-resident cell type. These cells contain both preformed and immunologically inducible TNF-a [64,65]. It is also remarkable, that TNF-a mRNA increases within 30–60 min [64]. Production of TNF-a is an important mechanism by which mast cells might influence physiological, immunological and

pathological processes [64]. Regarding these features, these cells might be interesting candidate cells for elevated TNF-a cytokine levels shortly after trauma. Taken together, we tested the potential of distinct cell types for TNF-a releasing capacity when carrying the TNF risk allele 308 A. Our findings indicate that high TNF-a levels detected in blood shortly after multiple trauma are not the result of a release from circulating blood leucocytes. We would have expected allelic imbalance of TNF-a secretion ex vivo if this would be the case. Candidate cellular sources of TNF-a in vivo after trauma and during sepsis include mast cells and other tissue-resident cells. Acknowledgments This work was supported by the National Genome Research Network (NGFN, Germany, Grant number NIE-S14T04). References [1] T.M.K. Menges, H. Hossain, S. Little, S. Tchatalbachev, F. Thierer, H. Hackstein, I. Franjkovic, T. Colaris, F. Martens, K. Weismuller, T. Langefeld, J. Stricker, G. Hempelmann, P. Vos, A. Ziegler, B. Jacobs, T. Chakraborty, G. Bein, Sepsis syndrome and death in trauma patients are associated with variation in the gene encoding tumor necrosis factor-a, Crit. Care Med. 36 (2008) 1456–1466. [2] B. Beutler, I. Milsark, A. Cerami, Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo, J. Immunol. 135 (1985) 3972–3977. [3] B. Beutler, D. Greenwald, J.D. Hulmes, M. Chang, Y.C. Pan, J. Mathison, R. Ulevitch, A. Cerami, Identity of tumour necrosis factor and the macrophagesecreted factor cachectin, Nature 8 (1985) 552–554. [4] D. Dubravec, D.R. Spriggs, J.A. Mannick, M.L. Rodrick, Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor a, Proc. Natl. Acad. Sci.USA 87 (1990) 6758–6761. [5] J. Djeu, D. Serbousek, D. Blanchard, Release of tumor necrosis factor by human polymorphonuclear leukocytes, Blood 76 (1990) 1405–1409. [6] P. Vassalli, The pathophysiology of tumor necrosis factors, Annu. Rev. Immunol. 10 (1992) 411–452. [7] K.-U. Belge, F. Dayyani, A. Horelt, M. Siedlar, M. Frankenberger, B. Frankenberger, T. Espevik, L. Ziegler-Heitbrock, The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF, J. Immunol. 168 (2002) 3536–3542. [8] L. Ziegler-Heitbrock, The CD14+ CD16+ blood monocytes: their role ininfection and inflammation, J. Leukocyte Biol. 81 (2007) 584–592. [9] F. Louis, G. Piron, R. Schaaf-Lafontaine, M. Mahieu, B. Groote, Tumour necrosis factor (TNF) gene polymorphism influences TNF-a production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans, Clin. Exp. Immunol. 113 (1998) 401–406. [10] M.W. van der Linden, T.W.J. Huizinga, D.-J. Stoeken, A. Sturk, R.G.J. Westendorp, Determination of tumour necrosis factor-a and interleukin-10 production in a whole blood stimulation system: assessment of laboratory error and individual variation, J. Immunol. Methods 218 (1998) 63–67. [11] R.P. D’Alfonso S, A polymorphic variation in a putative regulation box of the TNFA promoter region, Immunogenetics 39 (1994) 150–154. [12] T.S.N. Higuchi, S. Kamizono, A. Yamada, A. Kimura, H. Kato, K. Itoh, Polymorphism of the 5’-flanking region of the human tumor necrosis factor (TNF)-alpha gene in Japanese, Tissue Antigens 51 (1998) 605–6125. [13] A.M. Uglialoro, P.A. Pesavento, J.C. Delgado, F.E. McKenzie, J.G. Gribben, D. Hartl, E.J. Yunis, A.E. Goldfeld, Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-alpha gene promoter, Tissue Antigens 52 (1998) 359–367. [14] B.M. Brinkman, M.J. Giphart, A. Verhoef, E.L. Kaijzel, A.M.I.H. Naipal, M.R. Daha, F.C. Breedveld, C.L. Verweij, Tumor necrosis factor-a 308 gene variants in relation to majorhistocompatibility complex alleles and Felty’s syndrome, Hum. Immunol 41 (1998) 259–266. [15] A.G. Wilson, F.S. di Giovine, A.I.F. Blakemore, G.W. Duff, Single base polymorphism in the human tumour necrosis factor alpha (TNFa) gene detectable by Ncol restriction of PCR product, Hum. Mol. Genet. 1 (1992) 353. [16] B. Brinkman, E.L. Kaijzel, T.W. Huizinga, M.J. Giphart, F.C. Breedveld, C.L. Verweij, Detection of a C-insertion polymorphism within the human tumor necrosis factor alpha (TNFA), Hum. Genet. 96 (1995) 493. [17] A. Hamann, C. Mantzoros, A. Vidal-Puig, J.S. Flier, Genetic variability in the TNF-alpha promoter is not associated with type II diabetes mellitus, Biochem. Biophys. Res. Commun. 26 (1995) 833–839. [18] J.P. Mira, A. Cariou, F. Grall, C. Delclaux, M.R. Losser, F. Heshmati, C. Cheval, M. Monchi, J.L. Teboul, F. Riche, G. Leleu, L. Arbibe, A. Mignon, M. Delpech, J.F. Dhainaut, Association of TNF2, a TNF-a promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study, JAMA 282 (1999) 561– 568. [19] O. Appoloni, E. Dupont, M. Vandercruys, M. Andriens, J. Duchateau, J.L. Vincent, Association of tumor necrosis factor-2 allele with plasma tumor necrosis

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